Oligonucleotides that inhibit production of α-tumor necrosis factor

ABSTRACT

A sequence of anti-direction, anti-messenger RNA oligonucleotides of α-TNF characterized in that it possesses the structure of the formula ##STR1## wherein X is hydrogen, or a sequence of 1 to 17 oligonucleotides in free form, in alkylated form, in sulfurated form or in the form of a poly L-lysine derivative useful for stopping the production of α-TNF.

PRIOR APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.08/039,924 filed Mar. 29, 1993, now abandoned, which is a continuationof U.S. patent application Ser. No. 07/865,206 filed Apr. 8, 1992, nowabandoned, which is a continuation of U.S. patent application Ser. No.07/570,215 filed Aug. 21, 1990, now abandoned.

STATE OF THE ART

It is known that TNF (Tumor Necrosis Factor) secreted by macrophages isresponsible for absolute metabolic disasters that can create aninfectious endotoxinic shock. In seeking to stop the source of TNFproduction, the research of the sequences of anti-direction,anti-messenger RNA oligonucleotides which can thus stop the productionof TNF has been conducted. Pertinent prior art includes PCT aplicationWO.A. 806,625 and WO.A 8,604,606 and Nucleic Acids Research, Vol. 13 No.17 (1985), p. 6361 to 6373.

OBJECTS OF THE INVENTION

It is an object of the invention to provide novel sequences ofanti-direction, anti-messenger RNA oligonucleotides of α-TNF and theirpreparation.

It is another object of the invention to provide novel compositions anda novel method of stopping the production of α-TNF.

These and other objects and advantages of the invention will becomeobvious from the following detailed description.

THE INVENTION

The novel sequence of the invention of anti-direction, anti-messengerRNA oligonucleotides of α-TNF is characterized in that it possesses thestructure of the formula ##STR2## wherein X is hydrogen, or a sequenceof 1 to 17 oligonucleotides in free form, in alkylated form, insulfurated form or in the form of a poly-L-lysine derivative.

In formula I, the sequence of 1 to 17 oligonucleotides means anysequence of adenine and guanine (purine bases) and cytosine and thymine(pyrimidine bases). Alkylated form means alkylated derivatives with analkyl on the phosphate and especially methyl, ethyl or propyl whichgroups may be present on all or some of the phosphate groups. Sulfuratedforms means sulfurated derivatives of the phosphate, especially thioatesand dithioates which can be present on, some or all of the phosphategroups.

Examples of sequences of oligonucleotides of formula I are those whereinX is hydrogen or all or part of the sequence of the formula ##STR3## infree form, in alkylated form, in sulfurated form or in the form of apoly L-lysine derivative.

Among the latter, there are preferred the oligonucleotide sequences offormula I in which X is a hydrogen atom, a sequence of formula --CC orthe sequence of formula I_(A), in free form, in alkylated form, insulfurated form or in the form of a poly L-lysine derivative, andespecially the following sequences: ##STR4##

The novel process of the invention for the preparation ofoligonucleotides of formula I comprises immobilizing the firstnucleotide in position 3' of the chain on a support to form of theformula ##STR5## wherein S is the support, Z is a hydrocarbon of 2 to 20carbon atoms, B₁ is a purine or pyrimidine base corresponding to thefirst nucleotide, the amine function of which is protected and R is aprotective group, deblocking the hydroxyl in position 5' with an acidreagent to obtain a compound of the formula ##STR6## in which S, Z andB₁ have the above definitions, reacting the latter with the monomer ofthe second nucleotide of the formula ##STR7## in which R has the abovemeaning, B₂ is a purine or pyrimidine base corresponding to the secondnucleotide, the amine function of which is protected, R₁ is alkyl or--OR'₁ or SR'₁ wherein R'₁ is a protector group, and R₂ is a protectorgroup to obtain a product of the formula ##STR8## wherein S, R, R₁, B₁and B₂ have the above definitions, oxidizing the product of formula V toobtain a product of the formula ##STR9## wherein S, R, R₁, B₁, B₂ and Zhave the above definitions and X₁ is oxygen or sulfur, then, a new ringis produced with this product of formula VI as from the product offormula II and the monomer of a new nucleotide until the desired chainformation is obtained to obtain finally a product of the formula##STR10## wherein S, R, R₁, B₁, B₂, X₁ and Z have the above definitionsand B_(z) is the last nucleotide of the sequence, then deprotecting theoligonucleotide and separation from the support to obtain the product offormula I which is purified.

In the preferred operating conditions of the process, the deblocking ofthe hydroxyl in position 5' of the product of formula II is effectedwith an acid reagent such as acetic acid or di- or trichloroacetic acid,the coupling reaction of the product of formula III with the product offormula IV is activated with tetrazole in acetonitrile, the oxidation ofthe phosphite of formula V into the phosphate of formula VI is effectedwith iodine in solution of a water/lutidine/tetrahydrofuran mixture orin a water/pyridine/tetrahydrofuran mixture, the oxidation of thephosphite of formula V to obtain the sulfurated form VI is effected withsulfur in solution in a mixture of carbon sulfide, anhydrous pyridineand anhydrous triethylamine, at the end of the synthesis, thedeprotection of the terminal hydroxyl group in position 5' is effectedwith an acid such as acetic acid or di- or trichloroacetic acid and thedeprotection of the oligonucleotide of formula VII is effected withconcentrated ammonium hydroxide by moderately heating the reactionmedium.

For the operation of the process described above, the automaticsynthesizer "Applied Biosystems, Model 381A" is preferably used.

In the process of the invention, the support S is a solid support whichcan be constituted of silica or porous glass and can also be used thesupports described in European Patent No. 0,208,599.

According to the invention, Z is a hydrocarbon --(CH₂)_(n) -- in which nis an integer from 2 to 20. Products in which Z is --(CH₂)₂ -- arepreferably used.

In the formulae II to VII, the bases B₁, B₂ . . . . B_(z) are purine orpyrimidine bases, the amine functions of which are protected. Examplesof the protector groups are benzoyl or isobutyryl. In the case ofadonine and cytosine, benzoyl is preferably used. In the case ofguanine, isobutyryl is preferably used.

The protector group R of the hydroxyl function in position 5' is, forexample, trityl, monomethoxytrityl, dimethoxytrityl or pixyl.

The protector group of R'₁ of the hydroxyl functions of the phosphategroups can be for example, methyl, cyanoethyl, ortho orpara-chlorophenyl and the cyanoethyl group is preferably used. Theprotector group of R₂ can be alkyl such as methyl, ethyl, isopropyl ormorpholino or piperidino with isopropyl preferably used.

During the operation of the process, the fraction of the product offormula III which has not reacted is immediately converted into an esterto avoid the formation of a bad sequence for the following coupling.This reaction of "capping" is effected preferably with acetic anhydridein a lutidine/tetrahydrofuran mixture and is catalyzed by4-dimethylamino-pyridine or by methylimidazole.

The poly L-lysine derivatives of the oligonucleotide sequences can beprepared by a process characterized in that a product of formula##STR11## wherein S, Z, R, B₁ have the above definitions is reacted withproduct of formula IV to obtain a product of the formula ##STR12##wherein S, R, Z, R₁, B₁ and B₂ have the above definitions followed byoxidizing and eliminating the support and the activating group to obtaina product of the formula ##STR13## wherein R, R₁, B₁ and D₂ have theabove definitions, X₁ is oxygen or sulfur, the terminal ribose isoxidized then reacted with L-lysine in a reducing medium to obtain theproduct of the formula ##STR14## wherein R, R₁, B₁, B₂ and X₁ have theabove definitions and then the synthesis is continued as from theproduct of formula VI.

The poly L-lysine derivatives can be prepared by a process similar tothe process described by LEONETTI et al. GENE, Vol. 72 (1988), p, 323 to332.

The novel compositions for halting the production of -TNF are comprisedof an amount of a sequence of formula I sufficient to halt production ofα-TNF and an inert carrier or excipient. The compositions may be in theform of tablets, dragees, gelules, capsules, granules, suppositories,injectable solutions or suspensions and aerosols.

Examples of suitable excipients are talc, gum arabic, lactose, starch,magnesium stearate, cocoa butter, aqueous or non-aqueous vehicles, fattysubstances of animal or vegetable origin, paraffin derivatives, glycols,various wetting, dispersing or emulsifying agents, and preservatives.

The compositions, due to the fact that they possess the capability ofstopping the production of α-TNF by the cells by attaching themselvesspecifically to the target messenger RNA at the level of the sequence166 to 185! which is found astride the codon AUG, are useful for thetreatment of infectious endotoxinic shocks of all origins and of all thepathological conditions connected with a hypersecretion of α-TNF.

The novel method of the invention of halting the production of α-TNF inwarm-blooded animals, including humans, comprises administering towarm-blooded animals an amount of a sequence of formula I sufficient tohalt production of α-TNF. The sequence may be administered orally,rectally, parenterally or topically and the usual daily dose is 0.0133μg to 0.4 mg/kg depending on the condition treated, the specificsequence and the method of administration.

In the following examples there are described several preferredembodiments of the invention to illustrate the invention. However, itshould be understood that the invention is not intended to be specificembodiments.

EXAMPLE 1

Sequence (d) TCA TGG TGT CCT TTG CAG (SEQ ID NO:4) (18 "mer" anti-αTNF)in free form

An "Applied Biosystems Model 381 A" automatic synthesizer was used andthe apparatus was loaded with all the necessary reagents. The productsof formula IV were dissolved in acetonitrile under a light argonpressure which enabled them to be delivered automatically by the openingof electro-valves as and when required. The nucleotide sequence to besynthesized was recorded and the quantity of product on which onedesired to work was indicated. The final detritylation was optionallyprogrammed to recover the product of formula I in hydroxylated form orin tritylated form in position 5.

A small previously filled column containing the functionalized supportII was placed on the apparatus (CPG porous glass support). The synthesiswas then carried out from start to finish without manual intervention.The detritylation solutions were recovered automatically at each stagein a fraction collector coupled to the apparatus so that the yields ateach stage could be known.

Reagents

All the reagents used must be very pure. The 4 phosphoramidites offormula IV (B₂ =adenine, cytosine, guanine or thymine) were packed intosmall flasks and it was sufficient to add the necessary quantity ofsolvent before introducing them into the apparatus.

The other solutions introduced into the apparatus were the following: a)4% tetrazole in acetonitrile for the coupling activator, b) aceticanhydride/lutidine/tetrahydrofuran 1/1/8 on the one hand anddimethylaminopyridine/tetrahydrofuran 7/93 on the other hand mixed insitu for the "capping" reaction, c)Iodine/water/lutidine/tetrahydrofuran 3/2/2/93 for the oxidation of thephosphite of formula V into phosphate of formula VI, d) 2%trichloroacetic acid in dichloromethane for the detritylation reactionand e) pure acetonitrile for the dissolution of the phosphoramidites.

Method

The reaction was effected on 0.95 micromole and the product wasdetritylated in the apparatus at the end of the synthesis. The yield ofcrude product from the synthesis was 79%. The deblocking of the productof formula VII was effected in the following way:

1-28% ammonium hydroxide for one hour at ambient temperature and

2--saturated ammonium hydroxide at 60° C. for 5 hours.

Purification

1--Desalinization on NAP 10 (Pharmacia) Sephadex G50® column.

2--HPLC on Partisil 10 SAX® column, gradient of 50 to 99% of solvent Ain the mixture A+B over 20 minutes.

A=buffer of 0.3M phosphate/CH₃ CN 7/3 pH 6.2

B=buffer of 10⁻³ M phosphate /CH₃ CN 7/3 pH 6.2 supply rate 2 ml/min,

3--Desalinization on NAP 25 (Pharmacia) Sephadex G50® column.

Analysis of purified product

HPLC on Partisil 10 SAX® same conditions as above, RT=13.92 min.

HPLC on inverse phase microbondapack C18® gradient 8.4 to 15% of CH₃ CNin 10⁻² M triethylammonium acetate, pH 5.5, over 20 minutes, supply rate2 ml/min. TR=12.13 min.

Finally 0.125 micromolo of pure product was obtained in a yield of 13%.

EXAMPLE 2

Sequence (d) TCA TGG TGT CCT TTG CAG (SEQ ID NO:4) (18 "mer" anti-αTNF)all in thioate form

Using the procedure of Example 1, but replacing the oxidation cycleusing iodine by an oxidation stage using sulfur for 450 seconds, thedesired product mentioned was obtained. The solution ofiodine/water/lutidine/tetrahydrofuran was replaced by a solution of: 3 gof sulfur, 28.5 ml of carbon sulfide, 28.5 ml of anhydrous pyridine and3 ml of anhydrous triethylamine.

In the case of an oxidation with sulfur, it was indispensable to rinsethe column before and after oxidation, washing repeatedly with asolution of carbon sulfide in pyridine 1/1 to remove any sulfur residue.For the rest, the method remained unchanged. The Yield for synthesis on1.24 micromole was 56.5%.

Deblocking

1 28% ammonium hydroxide for one hour at ambient temperature

2--saturated ammonium hydroxide for one night at 50° C.

Purification

1--desalinization NAP 25 (Sephadex)® eluant H₂ O.

2--HPLC control microbondapack C18®RT=24.46

Gradient of 30 to 50% of A over 20 min., supply rate 2 ml/min.

A=triethylammonium acetate 10⁻² M/l., pH 5.5/CH₃ CN 7/3

B=triethylammonium acetate 10⁻² M/l., pH 5.5

Pure product yield was 40.3%

EXAMPLE 3

Sequence (d) TCA TGG TGT CCT TTG CAG CC (SEQ ID NO:3) (20 "mere"anti-αTNF) in free form

Using the procedure of Example 1, the desired product was obtained.

EXAMPLE 4

Sequence (d) TCA TGG TGT CCT TTG CAG CC TCA TGC TTT CAG TAG (SEQ ID NO:5) "mere" anti-αTNF) in free form

Using the procedure of Example 1, the desired product was obtained.

EXAMPLE 5

An injectable solute was prepared containing 10 micrograms of theproduct of Example 1 and sufficient sterile aqueous excipient for avolume of 2 ml.

EXAMPLE 6

Tablets were prepared containing 20 micrograms of the product of Example2 and sufficient excipient of lactoses, starch, talc and magnesiumstearate for a tablet of 100 mg.

EXAMPLE 7

An injectable solute was prepared containing 10 micrograms of theproduct of Example 3 and sufficient sterile aqueous excipient for avolume of 2 ml.

STUDY OF THE OLIGONUCLEOTIDE SEQUENCE PREPARED IN EXAMPLE 3

1--Biological test

1.1--Incubation of the human monocytes (4×10⁻⁶ cells) with

6 micromoles of anti-direction oligonucleotides in the

4 forms for 3 hours.

1.2--Then addition of the LPS (10 micrograms/ml) and the INF (5×10³μ/ml).

1.3--Incubation for 18 hours,

1.4--Taking up the culture supernatant.

1.5--Addition of this supernatant to the cells sensitive to lysis by TNF(L. 929) for 24 hours.

1.6--Measurement of the cellular viability by colorimetry with crystalviolet.

2--Immunological test

2.1--Incubation of the human monocytes (4×10⁶ cells) with 6 micromolesof anti-direction oligonucleotides in the 4 forms for 3 hours.

2.2--Next, addition of the LPS (10 micrograms/ml) and the INF (5×10³μ/ml) for the stimuation of the TNF production.

2.3--Incubation for 18 hours.

2.4--Taking up the culture supernatant.

2.5--Forming a coating with this culture supernatant.

2.6--Addition of anti-TNF antibody marked with iodine (for RIA) or wishan enzyme (for ELISA).

2.7--Incubation for 3 hours.

2.8--Readings

a) add the enzyme substrate (ELISA) and read the optical density.

b) measure the radioactivity (RIA).

2.9--Measure the quantity of TNF produced by the monocytes.

2.10--Calculate the value of inhibition of the TNF production.

3--RESULTS

Value of inhibition of the α-TNF production by the monocytes treatedwith anti-direction oligonucleotides was ##EQU1##

Various modifications of the products and method may be made withoutdeparting from the spirit or scope thereof and it is to be understoodthat the invention is intended to be limited only as defined in theappended claims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 5                                                  (2) INFORMATION FOR SEQ ID NO: 1:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19                                                                (B) TYPE: NUCLEIC ACID                                                        (C) STRANDEDNESS: SINGLE                                                      (D) TOPOLOGY: UNKNOWN                                                         (ii) MOLECULE TYPE: OLIGONUCLEOTIDE                                           (ix) FEATURE:                                                                 (A) NAME/KEY:                                                                 (B) LOCATION:                                                                 (C) IDENTIFICATION METHOD:                                                    (D) OTHER INFORMATION: IN THE SPECIFICATION X=N, X IS                         HYDROGEN, OR A SEQUENCE OF 1 TO 17 OLIGONUCLEOTIDES                           IN FREE FORM, IN ALKYLATED FORM, IN SULFURATED FORM                           OR IN THE FORM OF A POLY-L-LYSINE DERIVATIVE                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:                                      TCATGGTGTCCTTTGCAGN19                                                         (2) INFORMATION FOR SEQ ID NO: 2:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17                                                                (B) TYPE: NUCLEIC ACID                                                        (C) STRANDEDNESS: SINGLE                                                      (D) TOPOLOGY: UNKNOWN                                                         (ii) MOLECULE TYPE: OLIGONUCLEOTIDE                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:                                      CCTCATGCTTTCAGTAG17                                                           (2) INFORMATION FOR SEQ ID NO: 3:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20                                                                (B) TYPE: NUCLEIC ACID                                                        (C) STRANDEDNESS: SINGLE                                                      (D) TOPOLOGY: UNKNOWN                                                         (ii) MOLECULE TYPE: OLIGONUCLEOTIDE                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:                                      TCATGGTGTCCTTTGCAGCC20                                                        (2) INFORMATION FOR SEQ ID NO: 4:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: NUCLEIC ACID                                                        (C) STRANDEDNESS: SINGLE                                                      (D) TOPOLOGY: UNKNOWN                                                         (ii) MOLECULE TYPE: OLIGONUCLEOTIDE                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:                                      TCATGGTGTCCTTTGCAG18                                                          (2) INFORMATION FOR SEQ ID NO: 5:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35                                                                (B) TYPE: NUCLEIC ACID                                                        (C) STRANDEDNESS: SINGLE                                                      (D) TOPOLOGY: UNKNOWN                                                         (ii) MOLECULE TYPE: OLIGONUCLEOTIDE                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:                                      TCATGGTGTCCTTTGCAGCCTCATGCTTTCAGTAG35                                         __________________________________________________________________________

What is claimed is:
 1. A sequence of anti-direction, anti-messenger RNAoligonucleotides of α-TNF characterized in that it possesses thestructure of the formula

    5'TCA TGG TGT CCT TTG CAG-N 3'                             (SEQ ID NO:1)

wherein N is a hydrogen atom, or a sequence of the formula 5' CC 3', ora sequence of the formula 5' CC TCA TGC TTT CAG TAG 3', and wherein someof the phosphate groups are in free form, in alkylated form, in thiolateor dithiolate form, or in the form of poly-L-lysine derivative.
 2. Aprocess for the preparation of the oligonucleotide sequence of claim 1comprising immobilizing on a support S the first nucleotide in position3' of the chain to be synthesized of the formula ##STR15## wherein S isthe support, Z is a hydrocarbon of 2 to 20 carbon atoms, B₁ is a purineand pyrimidine base corresponding to the first nucleotide, the aminefunction of which is protected, and R is a protector group, deblockingthe hydroxyl in position 5' with an acid reagent to obtain a compound ofthe formula ##STR16## wherein S, Z and B₁ have the above definitions,reacting the latter with the monomer of the second nucleotide of theformula ##STR17## wherein R has the above definitions, B₂ is a purine orpyrimidine base corresponding to the second nucleotide, the aminefunction of which is protected, R₁ is alkyl or --OR'₁ or SR'₁ in whichR'₁ is a protector group and R₂ is a protector group to obtain a productof the formula ##STR18## wherein S, R, R₁, B₁ and B₂ have the abovedefinitions, oxidizing the latter product to obtain a product of theformula ##STR19## wherein S, R, R₁, B₁, B₂ and Z have the abovedefinitions and X₁ is oxygen or sulfur, then, reacting the product offormula VI with the product of formula IV and oxidizing, and repeatingfor addition of each new nucleotide until the desired chain formation isobtained to obtain a product of the formula ##STR20## wherein S, R, R₁,B₁, B₂, X₁ and Z have the above definitions and B_(z) is the lastnucleotide of the sequence, deprotecting the oligo-nucleotide,separation from the support and purifying the product of formula I. 3.The process of claim 2 wherein the deblocking of the hydroxyl inposition 5' of the product of formula II is effected with an acidreagent, the coupling reaction of the product of formula III with theproduct of formula IV is activated by tetrazole in acetonitrile, theoxidation of the phosphite of formula V into phosphate of formula VI iseffected with iodine in solution in a water/lutidine tetrahydrofuranmixture or in a water/pyridine/tetrahydrofuran mixture, the oxidation ofthe phosphite of formula V to obtain the sulfated form VI is effectedwith sulfur in solution in a mixture of carbon sulfide, anhydrouspyridine and anhydrous triethylamine, at the end of the synthesis, thedeprotection of the terminal hydroxyl group in position 5' is effectedwith an acid, the deprotection of the oligonucleotide of formula VII iseffected with concentrated ammonium hydroxide by moderately heating thereaction medium.
 4. The process of claim 3 wherein the acid deblockingand deprotecting agent is acetic acid or dichloroacetic acid ortrichloroacetic acid.
 5. An oligonucleotide sequence of claim 1 whereinN is CC of the formula ##STR21## wherein all or some of the phosphategroups are in free form, in alkylated form, in thiolate or dithiolateform, or in the form of a poly L-lysine derivative.
 6. Anoligonucleotide sequence of claim 1 wherein N is zero of the formula##STR22## wherein all or some of the phosphate groups are in free form,alkylated form, in thiolate or dithiolate form, or in the form of a polyL-lysine derivative.
 7. An oligonucleotide sequence of claim 1 wherein Nis the sequence SEQ ID No. 2 of the formula ##STR23## wherein all orsome of the phosphate groups are in free form, alkylated form, inthiolate or dithiolate form, or in the form of a poly L-lysinederivative.
 8. A composition for reducing the production of α-TNFcomprising an effective amount of a sequence of claim 1 to reduce theproduction of α-TNF and an inert pharmaceutical carrier.
 9. A method ofreducing the production of α-TNF in a cultured human cell comprisingproviding to a cultured human cell an amount of the oligonucleotide ofclaim 1 which inhibits production of α-TNF by the cell.